Agonist binding to GPCRs causes rapid phosphorylation of the activated receptors by GPCR kinases. This process promotes the recruitment of members of the arrestin protein family (beta-arrestin-1 and -2) to the activated receptors, disrupting receptor/G protein coupling and promoting GPCR internalization by targeting the receptors to clathrin-coated pits. However, during the past 10-15 years, many studies have demonstrated that beta-arrestins can also act as signaling molecules in their own right. This observation is not only of theoretical interest but also of potential clinical relevance. Despite recent advances in understanding the molecular mechanisms of beta-arrestin function, the potential interplay of heterotrimeric G proteins with beta-arrestin-mediated processes remains to be investigated. Generation of biased M3 muscarinic receptors Activation of the M3 muscarinic receptor (M3R) leads to the activation of G proteins of the Gq family. The activated receptor also recruits beta-arrestin-1 and -2 (barr1 and barr2, respectively) which mediate M3R desensitization. In addition, binding of barr1/2 to the activated M3R leads to conformational changes that allow barr1 and barr2 to act as scaffolding proteins for various intracellular signaling cascades. To gain insight into the contribution of barr1/2 signaling to M3R function, we generated two biased mutant M3Rs. One of the mutant receptors couples to Gq but is unable to recruit beta-arrestins. The other mutant receptor is no longer capable of activating Gq but retains the ability to interact with barr1/2. We are planning to carry out structural and functional studies with these two mutant M3Rs to gain insight into the role of beta-arrestins in M3R signaling in vitro and in vivo.